This presentation will help the students for rDNA technology-related topic.

1. Recombinant DNA Technology

2. Introduction
 Based on the concept of gene recombination.
 Encompasses a number of experimental protocols leading to the transfer of
genetic information(DNA) from one organism to another. Involves the
manipulation of genetic material(DNA) to achieve the desired goal in a pre-
determined way.
 rDNA technology has its roots in the experiments performed by Boyer & Cohen.
 The procedure has been used to change DNA in living organisms and may have
even more practical uses in the future.
 It is an area of medical science that is just beginning to be researched in a
concerted effort.

3. Basic Principle of rDNA technology
 Generation of DNA fragments & selection of the desired piece of DNA.
 Insertion of the selected DNA into a cloning vector to create a rDNA or chimeric DNA.
 Introduction of the recombinant vectors into host cells.
 Multiplication & selection of clones containing the recombinant molecules.
 Expression of the gene to produce the desired product.

4. Process of rDNA Technology
 DNA molecules that are constructed with DNA from different sources are called
recombinant DNA molecules.
 Recombinant DNA molecules are created in nature more often than in the
laboratory; for example, every time a bacteria phage or eukaryotic virus infects
its host cell and integrates its DNA into the host genome, a recombinant is
created.
 Occasionally, these viruses pick up a fragment of host DNA when they excise
from their host’s genome; these naturally occurring recombinant DNA molecules
have been used to study some genes.

5. Process of rDNA Technology
 Six steps of Recombinant DNA
1. Isolating (vector and target gene)
2. Cutting (Cleavage)
3. Joining (Ligation)
4. Transforming
5. Cloning
6. Selecting (Screening)

6. 1. Isolating (vector and target gene)
 First step in rec DNA technology is the selection of a DNA segment of interest which
is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA
segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned
DNA.
 A cloning vector is a self-replicating DNA molecule, into which the DNA
insert is to be integrated. A suitable cloning vector is selected in the next step
of rec DNA technology. Most commonly used vectors are plasmids and
bacteriophages.

7. 2. Cutting (Cleavage)
 Cleavage of DNA at particular sequences. As we will see, cleaving DNA to
generate fragments of defined length, or with specific endpoints, is crucial to
recombinant DNA technology. The DNA fragment of interest is called insert
DNA. In the laboratory, DNA is usually cleaved by treating it with commercially
produced nucleases and restriction endonucleases.

8. 3. Joining (Ligation)
 A recombinant DNA molecule is usually formed by cleaving the DNA of interest
to yield insert DNA and then ligating the insert DNA to vector DNA
(recombinant DNA or chimeric DNA). DNA fragments are typically joined using
DNA ligase (also commercially produced). Eg. T4 DNA Ligase

9. 4.Transforming
 Introduction of recombinant DNA into compatible host cells. In order to be propagated,
the recombinant DNA molecule (insert DNA joined to vector DNA) must be introduced
into a compatible host cell where it can replicate. The direct uptake of foreign DNA by
a host cell is called genetic transformation (or transformation). Recombinant DNA can
also be packaged into virus particles and transferred to host cells by transfection.

10. 5. Cloning
 Cloning vectors allow insert DNA to be replicated and, in some cases, expressed
in a host cell. The ability to clone and express DNA efficiently depends on the
choice of appropriate vectors and hosts.

11. 6. Selecting (Screening)
 Vectors usually contain easily scored genetic markers, or genes, that allow the
selection of host cells that have taken up foreign DNA. The identification of a
particular DNA fragment usually involves an additional step—screening a large
number of recombinant DNA clones. This is almost always the most difficult
step.

12. Process of rDNA Technology
Source: https://www.biologyexams4u.com/2013/10/steps-in-recombinant-dna-technology.html#.XRzVB-gzbIU

13. Applications of rDNA technology
 Manufacture of proteins/hormones Interferon, plasminogen activating factor,
blood clotting factors, insulin, growth hormone.
 AIDS test: Has become simple & rapid.
 Diagnosis of molecular diseases: sickle cell anaemia, thalassaemia, familial
hypercholesterolaemia, cystic fibrosis.
 Prenatal diagnosis: DNA from cells collected from amniotic fluid, chorionic
villi.
 Gene Therapy: This is achieved by cloning a gene into a vector that will readily
be taken up & incorporated into genome of a host cell. ADA deficiency has been
successfully treated.

14. Applications of rDNA technology
 Application in Agriculture: Genetically engineered plants are developed to
resist draught & diseases. Good quality of food & increased yield of crops is also
possible.
 Industrial Application: Enzymes-use to produce sugars, cheese, detergents.
Protein products, used as food additives, increases nutritive value, besides
imparting flavour.
 Application in forensic medicine: The restriction analysis pattern of DNA of
one individual will be very specific(DNA fingerprinting),but the pattern will be
different from person to person. Helps to identify criminals & to settle disputes of
parenthood of children.

15. Questions
1. A recombinant DNA molecule is produced by which of the following…
a) joining of two DNA fragments b) joining of two or more DNA fragments
c) both a and b d) joining of two or more DNA fragments originating from different organisms
2. The gene formed by the joining of DNA segments from two different sources are termed as…
a) recombinant gene b) joined gene c) both a and b d) chimaeric gene
3. Which of the following enzyme is used to cut DNA molecule in recombinant DNA technology?
a) ligase b) phosphatase c) ribonuclease d) restriction enzymes
4. Restriction enzymes are also termed as…
a)biological scissors b) molecular scalpels c) molecular knives d) all of these
5. Which of the following created the first rDNA molecule?
a) Nathan, Arber and Smith b) Watson, Crick and Wilkins c) Boyer and Cohen d) Paul Berg

16. Questions
6. The DNA molecule to which the gene of insert is integrated for cloning is called as…
a) carrier b) transformer c) vector d) none of these
7. The DNA segment to be cloned is called as…
a) gene segment b) DNA fragment c) DNA insert d) all of these
8. The first successful transformation of rDNA molecule into a bacterium was carried out by which of the
following…
a) Nathan, Arber and Smith b) Watson, Crick and Wilkins c) Boyer and Cohen d) Paul Berg
9. The plasmid used by Cohen and Boyer for their transformation experiment was which of the following
a) pSC 101 b) PUC 17 c) pBR 322 d) E.coli plasmids
10. The mechanism of intake of DNA fragments from the surrounding medium by a cell is termed as..
a) transformation b) transduction c) both a and b d) conjugation

17. Questions
11. Which of the following restriction enzymes produce blunt ends?
a) SaII b) EcoRV c) XhoI d) HindIII
12.Restriction enzymes are named for which of the following?
a) the person who discovered b) the bacterium they are derived from
c) the viral DNA that they attack d) none of the above
13. First DNA sequences were obtained in which of the following?
a) 1930s b)1920s c)1970s d)1980s
14. HaeIII and AluI are restriction enzymes that cut straight across double helix producing which kind of
sequence?
a) sticky ends b) blunt ends c) Codons d) Anticodons
15. Gene cloning refers to which of the following?
a) production of large number of copies of the gene being cloned b) production of asexual progeny from a single
individual or a cell c) both a and b d) none of these

18. Answers
1. d
2. d
3. d
4. a
5. d
6. c
7. c
8. c
9. a
10. a

19. Answers
11. b
12. b
13. c
14. b
15. a

20. Thanks….